1. Hemostasis

2. Hemostasis („hemo”=blood; sta=„remain”) is the stoppage of bleeding, which is vitally important when blood vessels are damaged.
Following an injury to blood vessels several actions may help prevent blood loss, including:
Formation of a clot

3. Local vasoconstriction
is due to local spasm of the smooth muscle (symp. reflex)
can be maintained by platelet vasoconstrictors

4. Formation of platelet aggregate
Injured blood vessel releases ADP, which attracts platelets (PLT)
PLT comming in contact with exposed collagen release: serotonin, ADP, TXA2, which accelerate vasoconstriction and causes PLT to swell and become more sticky

5. The micrograph shows activated platelets adhering to some damaged cells

6. Formation of blood clot
In the formation of the clot, an enzyme called thrombin converts fibrinogen into insoluble protein, fibrin
Fibrin aggregates to form a meshlike network at the site of vascular damage

7. Coagulation mechanism is composed of an extrinsic and intrinsic pathway, which eventually merge into one
The intrinsic system is more complex and present only in „higher” life forms (e.g. birds and reptiles possess only extrinsic system)
The complex sequence of events that produce fibrin are divided into three stages

8. Stage I: Formation of prothrombin activator
Extrinsic pathway:
1. When blood comes in contact with injured tissue – tissue thromboplastin (F III) interacts with proconvertin (F VII), and Ca2+ activating Stuart factor (F X).
Ca2+
Christmas factor
Anti hemophilic factor
Stuart factor
Stage I: Formation of prothrombin activator

9. Stage I: Formation of prothrombin activator
Intrinsic pathway:
2. Exposed collagen activates Hageman factor (F XII). Activated F XII activates plasma enzyme – plasma thromboplastin antecedent (PTA; F XI, which in the presence of Ca 2+ activates Christmas factor (F IX). F IX interacts with antihemophilic factor (F VIII), Ca 2+ to form a complex that activates Stuart factor (F X).
Ca2+
Christmas factor
Anti hemophilic factor
Stuart factor
Stage I: Formation of prothrombin activator

10. Stage I: Formation of prothrombin activator
3. Common pathway:
Activated F X in the presence of Ca 2+ forms complexes with accelerin (F V) to form prothrombin activator
Ca2+
Christmas factor
Anti hemophilic factor
Stuart factor
Stage I: Formation of prothrombin activator

12. Stage II: conversion of prothrombin to thrombin
Ca2+
Prothrombin – inactive precursor of enzyme thrombin
In the presence of prothrombin activator and Ca2+ prothrombin is converted to thrombin
Thrombin itself increases its own rate of formation (positive feedback mechanism)

13. Stage III: conversion of fibrinogen to fibrin
fibrin-stabilizing factor
Fibrinogen – plasma protein produced by the liver
Thrombin converts fibrinogen to fibrin
Thrombin also activates fibrin-stabilizing factor (F XIII), which in the presence of Ca2+, stabilizes the fibrin polymer through covalent bonding of fibrin monomers

14. Calcium ions
Are required for promotion and acceleration of almost all blood clotting reactions
Except: activation of XII and XI (intrinsic mechanism)
Ca2+

15. Stuart factor Christmas factor Anti- hemophilic factor
Ca2+
Christmas factor
Anti hemophilic factor
Stuart factor
Ca2+
Fibrin-stabilizing factor

18. Fibrinolysis

19. Clot Dissolution Plasminogen Plasmin Fibrin soluble fragments
Plasmin is formed from plasminogen - enzyme called activator (e.g. enzymes from urine, tears, saliva or bacterial enzyme streptokinase)
Plasmin as an enzyme is involved in breaking down fibrin into soluble fragments (fibrinolysis)
Activator (e.g. t-PA)
Plasminogen Plasmin
Fibrin soluble fragments
Plasminogen may be produced by eosinophils

21. Hirudo medicinalis produce Hirudin that inhibits Thrombin
Anticoagulants
Hirudo medicinalis produce Hirudin that inhibits Thrombin

22. Anticoagulants
Although tissue breakdown and platelets destruction are normal events in the absence of trauma, intravascular clotting does not usually occur because:
the amounts of procoagulants released are very small
natural anticoagulants are present (Antithrombin III, Heparin, Antithromboplastin, Protein C and S, fibrin fibers)

23. Natural anticoagulants
Antithrombin III – inhibits factor X and thrombin
Heparin from basophils and mast cells potentiates effects of antithrombin III (together they inhibit IX, X, XI, XII and thrombin)
Antithromboplastin (inhibits „tissue factors” – tissue thromboplastins)
Protein C and S – activated by thrombin; degrade factor Va and VIIIa

24. Abnormalities of hemostasis

25. Severe reduction in the number of PLTs - thrombocytopenia
this causes spontaneous bleeding as a reaction to minor trauma
in the skin - reddish- purple blotchy rash
it may result from:
decreased production (toxins, radiation, infection, leukemias)
increased destruction (autoimmune processes)
increased PLTs consumption (DIC)
Thrombocytopenia
Hemorrhagic spots (petechiae)

26. Thrombocytopenia Lethal when PLTs<10G/L
Bleeding occurs when PLTs<50G/L
Norm: G/L

27. Hepatic failure Most of the clotting factors are formed in the liver
Subconjunctival hemorrhage

28. Disseminated intravascular coagulation (DIC)
Widespread coagulation  thrombosis in small blood vessels  increased fibrinolysis, and depletion of coagulating factors  generalized bleeding
It may result from:
bacterial infections (endothelial damage)
disseminated cancers (release of procoagulants)
complications of pregnancy
severe catabolic states
Disseminated cervical cancer metastases (PET imaging)

29. Hemophilia A (lack of F VIII) and B (lack of F IX) are transmitted genetically and affect only males. Females carry the gen but do not show symptoms.
Von Willebrand’s disease – loss of large component of fVIII

30. Hemophilia A (lack of F VIII; 85%)
Spontaneous or traumatic subcutaneous bleeding
Blood in the urine
Bleeding in the mouth, lips, tongue
Bleeding to the joints, CNS, gastrointestinal tract
Mild hemophilia after injection in buttock

31. Son of the last Tsar of Russia – Aleksy Romanow suffered from Hemophilia A

32. Tests of coagulation

33. "Intrinsic" and "extrinsic" coagulation pathways
Activated Partial Thromboplastin Time
N: sec
Prothrombin Time
N: 9.9 – 13 sec

34. Prothrombin time (PT) test – norm 11 -15 sec evaluates extrinsic system (VII, X, V, II, fibrinogen)
prolonged PT indicates a deficiency in any of factors VII, X, V, prothrombin (factor II), or fibrinogen (factor I).
Prolonged PT:
-   a vitamin K deficiency (vitamin K is a co-factor in the synthesis of functional factors II (prothrombin), VII, IX and X)
-    liver disease
Warfarin therapy
DIC
excesive heparin

35. International Normalised Ratio (INR)
Therapeutic interval: Therapeutic interval for oral anticoagulant therapy:
Application: Monitoring oral anticoagulant therapy (eg. Warfarin);
note that heparin will not prolong INR (heparinase is included within the INR reagent)!!!!!!!!!!!!! For heparin therapy we monitor aPTT and/or aPTT ratio
The result for the PT is expressed as a ratio (prothrombin clotting time for patient plasma divided by time for control plasma);
Correction factor (International Sensitivity Index) is applied to the prothrombin ratio and the result issued as INR.

36. Most common case of prolonged aPTT – heparin!!!
Activated Partial Thromboplastin Time test (aPTT) – norm: s; evaluates intrinsic system (VIII, IX, XI, XII, X, V, II, fibrinogen)
an isolated prolongation of the aPTT (PT normal) suggests deficiency of factor VIII, IX, XI or XII
prolongation of both the APTT and PT suggests factor X, V, II or I (fibrinogen) deficiency, all of which are rare
aPTT is normal in factor VII deficiency (PT prolonged) and factor XIII deficiency
Most common case of prolonged aPTT – heparin!!!

37. Thrombin time (TT) – norm: 14-15 sec
Prolonged TT:
Heparin (much more sensitive to heparin than aPTT)
Hypofibrinogenemia

38. Selected causes of abnormal coagulation tests
Partial Thromboplastin Time (aPTT)
Prothrombin Time (PT)
Thrombin Time (TT)
Bleeding Time (BT)
Factor deficiency (except VII)
VII, X, V, II, fibrinogen deficiency
Low or absent fibrinogen
Thrombocytopenia
Antibodies to clotting factors
Antibodies
Dysfibrinogenemia, hypofibrinogenemia
Von Willebrand’s disease
Heparin
Warfarin; Vit K defficiency (mild to severe)
Drugs (Aspirin, NSAIDs, high dose penicillins, etc.)
Excessive Warfarin
Excessive Heparin
Cirrhosis, Uremia, PLTs dysfunction

39. "Intrinsic" and "extrinsic" coagulation pathways
Activated Partial Thromboplastin Time
N: sec
N: 9.9 – 13 sec

40. Whole blood clotting time
The time taken for blood to clot mainly reflects the time required for the generation of thrombin
The surface of the glass tube initiates the clotting process. This test is sensitive to the factors involved in the intrinsic pathway
The expected range for clotting time is 4-10 mins.

41. Whole blood clotting time – procedure:
Clean the tip of the finger with an alcohol
Prick the finger tip with an automatic lancet
Note the time when blood first appears on the skin
Touch the tube to the drop of blood
Break gently 1cm of the tube at the end of 2 min, and every 30 sec these after
When fibrin is formed between the two broken pieces of tube the coagulation or clotting time is noted

42. Bleeding time
This is a test that measures the speed in which small blood vessels close off (the condition of the blood vessels and platelet function)
This test is useful for detecting bleeding tendencies
The bleeding stops within 1 to 9 minutes. This may vary from lab to lab, depending on how the test is measured
Using the ear lobe method, a normal bleeding time is between 1 and 4 minutes.

43. Bleeding time – procedure:
Clean the earlobe with an alcohol
Prick the earlobe with an automatic lancet
Note the time when blood first appears on the skin
After half a minute (30sec) place the edge of the filter paper on the top of the drop of blood.
Perform the operation at half minute (30 sec) interval
The end point or bleeding time is the first half minute when no blood is seen on the filter paper.

44. Abnormal Bleeding Time
Prolonged bleeding time may indicate:
A vascular (blood vessel) defect
A platelet function defect (see platelet aggregation)
platelets count defect (low platelets)
Drugs that may increase times include dextran, indomethacin, and salicylates (including aspirin).

45. http://www. medicine. mcgill

47. The new model of haemostasis

48. 1. Initiation phase
Injury of vessels wall leads to contact between blood and subendothelial cells
Tissue factor (TF) is exposed and binds to FVIIa or FVII which
is subsequently converted to FVIIa
The complex between TF and FVIIa activates FIX and FX
FXa binds to FVa on the cell surface

49. 2. Amplification phase
The FXa/FVa complex converts small amounts of prothrombin into
thrombin
The small amount of thrombin generated activates FVIII, FV, FXI
and platelets locally.
FXIa converts FIX to FIXa
Activated platelets
bind FVa, FVIIIa
and FIXa

50. 3. Propagation phase
The FVIIIa/FIXa complex activates FX on the surfaces of activated platelets
FXa in association with FVa converts large amounts of prothrombin into thrombin creating
“thrombin burst”.
The “thrombin burst” leads to the formation of a stable fibrin clot.

51. Summary:
Haemostasis starts with the interaction between TF and FVIIa on the surface of subendothelial cells.
The small amount of thrombin generated during the amplification phase activates platelets locally on whose surface the subsequent reactions take place.
The resulting thrombin burst results in the formation of a stable clot.

52. NovoSeven® Mode of Action
Eptacog alfa (activated)
Tissue factor (TF)/FVIIa,
or TF/rFVIIa interaction, is necessary to initiatiate haemostasis
At pharmacological concentrations rFVIIa directly activates FX on the surface of locally activated platelets.
This activation will initiate the ”thrombin burst”
independently of FVIII and FIX. This step is independent of TF.
The thrombin burst leads to the formation of a stable clot

53. Conclusion:
• In high doses rFVIIa binds to the surface of the locally activated platelets where it leads to the formation of a ”thrombin burst”

54. Prescribing Information
NovoSeven® Eptacog alfa (activated) Abbreviated Prescribing Information: NovoSeven [Recombinant Coagulation Factor VIIa (rFVIIa)] Presentation: Powder for injection with accompanying solvent for reconstitution (Water for Injections). Available in packs containing 1.2, 2.4 or 4.8 mg rFVIIa. Uses: Treatment of bleeding episodes and prevention of bleeding during surgery or invasive procedures in patients with: - congenital haemophilia with inhibitors to coagulation factors VIII or IX > 5 BU or who are expected to have a high anamnestic response to FVIII or FIX. - acquired haemophilia - congenital FVII deficiency - Glanzmann’s thrombasthenia with antibodies to GP IIb-IIIa and/or HLA, and with past or present refractoriness to platelet transfusion. Dosage: The rFVIIa is dissolved in the accompanying solvent before use. After reconstitution the solution contains 0.6 mg rFVIIa/ml. Administer by intravenous bolus injection over 2-5 minutes; must not be mixed with infusion solutions or given in a drip. Haemophilia A or B with inhibitors or acquired haemophilia Initial dose of 90g per kg body weight. Duration of, and interval between, repeat injections dependent on severity of haemorrhage or procedure/surgery performed. For mild to moderate bleeding episodes (including ambulatory treatment): 1-3 doses at 3 hour intervals (90g per kg b.w.) to achieve haemostasis, with additional dose to maintain haemostasis. Duration of ambulatory treatment should not exceed 24 hours. For serious bleeding episodes, initial dose 90g per kg. b.w.; dose every two hours until clinical improvement. If continued therapy indicated, dosage interval can be increased successively. Major bleeding episode may be treated for 2-3 weeks or longer if clinically warranted. For invasive procedures/surgery administer initial dose of 90g per kg. b.w. immediately before the procedure. Repeat dose at 2-3 hour intervals for first hours. In major surgery continue dosing at 2-4 hour intervals for 6-7 days. Dosage interval may then be increased to 6-8 hours for further 2 weeks. Treatment may be up to 2-3 weeks until healing has occurred. Factor VII deficiency For bleeding episodes and for invasive procedures/surgery administer 15-30µg per kg b.w. every 4-6 hours until haemostasis achieved. Adapt dose and frequency to individual. Glanzmann’s thrombasthenia For bleeding episodes and for invasive procedures/surgery administer 90µg (range µg) per kg b.w. every 2 hours ( hours). At least three doses should be administered to secure effective haemostasis. For patients who are not refractory platelets are first line treatment. Contra-indications: Known hypersensitivity to active substance, excipients, or to mouse, hamster or bovine protein. Precautions: For severe bleeds NovoSeven should only be administered in hospitals specialised in the treatment of patients with coagulation factor VIII or IX inhibitors or in close collaboration with a physician specialised in treatment of haemophilia. Ambulatory treatment should not exceed 24 hours. Possibility of thrombogenesis or induction of DIC in conditions in which tissue factor could be expected in circulating blood, e.g. advanced atherosclerotic disease, crush injury, septicaemia, or DIC. Since NovoSeven may contain trace amounts of mouse, bovine and hamster proteins there is a remote possibility of the development of hypersensitivity. Monitor FVII deficient patients for prothrombin time and FVII coagulant activity; suspect antibody formation if FVIIa activity fails to reach expected level or bleeding not controlled with recommended doses. Avoid simultaneous use of prothrombin complex concentrates, activated or not. Use in pregnancy: Only administer to pregnant women if clearly needed. Not known if excreted in human milk; exercise caution when administering NovoSeven to nursing women. Side Effects: Adverse reactions (serious and non-serious) reported during post-marketing period: Rare (>1/10,000, <1/1,000): Lack of efficacy. Very rare <1/10,000): Coagulopathic disorders such as increased D-dimers and consumptive coagulopathy; myocardial infarction; nausea; fever; pain, especially at injection site; increase of ALT, ALP, LDH and prothrombin levels; cerebrovascular disorders including cerebral infarction and cerebral ischaemia; skin rashes; venous thrombotic events; haemorrhage.
Serious adverse reactions include: Arterial thrombotic events (such as myocardial infarction or ischaemia, cerebrovascular disorders and bowel infarction); venous thrombotic events (such as thrombophlebitis, deep vein thrombosis and pulmonary embolism). In the vast majority of cases patients were predisposed to such events. No spontaneous reports of anaphylactic reactions, but patients with a history of allergic reaction should be carefully monitored. No reports of antibodies against FVII in haemophilia A or B patients. Isolated cases of FVII-deficient patients developing antibodies against FVII reported after treatment with NovoSeven. These patients previously treated with human plasma and/or plasma derived FVII. Monitor FVII deficient patients for FVII antibodies. One case angioneurotic oedema reported in patient with Glanzmann’s thrombasthenia after administration of NovoSeven. Marketing Authorisation numbers: NovoSeven 60 KIU EU/1/96/006/001 NovoSeven 120 KIU EU/1/96/006/002 NovoSeven 240 KIU EU/1/96/006/003 Legal Category: POM Basic NHS Price: NovoSeven 1.2 mg £ NovoSeven 2.4 mg £ NovoSeven 4.8 mg £ Further information: Full prescribing information can be obtained from: Novo Nordisk Limited Broadfield Park Brighton Road Crawley West Sussex RH11 9RT Tel: Fax: Date of preparation: May 2004 Ref N7/03/039a

55. A 35-year-old man complains of chronic physical fatigue, which began 3-4 weeks ago. He said he felt tired all of the time even through his occupation as a software developer was mentally but not physically demanding. He breathed comfortably at rest but, when he exerted himself, he experienced difficulty in breathing and had hard time catching his breath. He also complained of „more than usual” mental fatigue, confessing an increasing inability to concentrate and focus his attention on tasks at hands. Colleagues noticed his pallor and his inattentiveness at brainstorming sessions and suggested he reschedule his annual physical examination for an earlier date. He complained of vague abdominal pain and sense of abdominal fullness. His appetite was depressed, and he thought perhaps his physical and mental symptoms were caused by poor diet. However, attempts to increase eating resulted in nausea. His stools, he said, were sometimes loose and tarry. Eventually, increased heart palpitations and chest pain made him seek medical advice

56. Laboratory findings revealed the following:
Laboratory test
Patient
Normal
RBC (red blood cell count)
3.5 T/L
T/L
HCT (hematocrit ratio)
28%
40-52%
Hb (hemoglobin)
8.0g/dL
13-17g/dL
MCV (mean corpuscular volume)
70fL
78-95fL
MCH (mean corpuscular hemoglobin)
22.8pg
29pg
MCHC (mean corpuscular hemoglobin concentration)
34%

57. Case history questions:
What general medical condition is suggested by the person’s symptoms?
What fundamental change in function of blood related to the red blood cells could simultaneously affect the function of several systems (cardiovascular, respiratory, gastrointestinal, and others)?
What specific diagnosis is supported by the laboratory findings?
How could the stool be related to the laboratory findings?

58. Answers:
Anemia
A reduction in oxygen-carrying capacity of the blood and thus a reduction in the delivery of oxygen to various body tissues
An iron defficiency anemia
Most cases of iron-defficiency anemia result from internal blood loss Dark, tarry loose stools suggest bleeding from the gastrointestinal tract and warrant further tests to determine the exact cause